This invention relates to a method of detecting and quantifying glycosylated proteins. More particularly, the invention relates to an immunoassay for detecting and quantitating proteins that are glycosylated on their amino-terminal (N-terminal) amino acid. One such protein is hemoglobin A.sub.1c (HbA.sub.1c), and the invention is particularly directed to immunoassays for detecting and quantitating HbA.sub.1c. The invention also relates to antibodies specific for reduced glycosylated N-terminal amino acids that are used in the immunoassays of the invention and to immunogens and methods for making these antibodies.
The in vivo glycosylation (also called "glycation") of proteins may occur either enzymatically or non-enzymatically. In non-enzymatic glycosylation, a sugar is covalently coupled to the epsilon amino groups of available lysine residues or to the alpha amino group of accessible amino terminal (N-terminal) amino acids of the protein.
Non-enzymatic glycosylation of proteins with glucose proceeds in two stages. First, glucose combines with the amino group of the lysine or of the N-terminal amino acid to form an aldimine compound (a Schiff base). This reaction is reversible, and dissociation to unmodified protein and glucose readily occurs. Next, the aldimine intermediate is converted to a stable ketoamine derivative (1-deoxyfructose) by the Amadori rearrangement. Finally, over a period of weeks, the free carbonyls of the ketoamine derivatives form crosslinks between the glycosylated protein and adjacent proteins, and the resulting aggregates are called advanced glycosylation products.
Non-enzymatic glycosylation occurs in normal mammals and to a much greater extent in diabetic patients. Patients afflicted with diabetes are incapable of metabolizing glucose in a conventional manner resulting in increased amounts of glucose in their blood and urine. In diabetic and normal individuals, glucose has been shown to bind non-enzymatically to the amino groups of many proteins, including hemoglobin, collagen, albumin, lens crystallins, fibrinogen, lipoproteins, ferritin, myelin, transferrin, and immunoglobulins, although such glycosylation occurs to a greater extent in diabetics than in normals.
The quantitative measurement of glycosylated proteins in diabetes is clinically important for two reasons. First, the measurement of glycosylated protein levels allows for the monitoring of blood glucose levels over an extended time period and allows the assessment of diabetic metabolic control. The time period over which mean blood glucose concentrations may be determined is largely dictated by the length of time a given assayed protein normally remains in the circulation. Since transferrin and human serum albumin are normally present in the circulation for a short to intermediate time (the half-life of transferrin is about 8 days, and the half-life of albumin is about 10-14 days), the measurement of the glycosylated forms provides a means of assessing short to intermediate glycemia. Hemoglobin is normally present in the circulation for a long time (the half-life of hemoglobin being about 2 to 3 months), and the measurement of glycosylated hemoglobin provides a means of assessing long term glycemia.
Second, non-enzymatic glycosylation has been implicated in the development of chronic diabetic complications, including accelerated cataract formation (due to advanced glycosylation products of crystallin lens protein) and atherogenesis (due to lipoprotein entrapment on arterial walls) contributing to the narrowing of blood vessels of the heart, brain, eyes, kidneys, and periphery. The measurement of glycosylated proteins and their advanced products may, therefore, be of prognostic value in the pathological sequelae involved in chronic hyperglycemia.
The main component of human hemoglobin (about 80-90% of the total hemoglobin) is HbA.sub.o. It has a amino acids. Preparations of HbA.sub.o isolated by ion tetrameric structure comprising two alpha and two beta chains. HbA.sub.o is not glycosylated on its N-terminal exchange chromatography have been shown to contain a small proportion of HbA.sub.o molecules glycosylated on the available epsilon amino groups of lysines.
HbA.sub.1a1, HbA.sub.1a2, HbA.sub.1b and HbA.sub.1c are minor components of human hemoglobin identical in structure to HbA.sub.o, except for the presence of a sugar residue covalently attached to the N-terminal valine residue of the beta chain. The sugar residues attached to the valine residues of HbA.sub.1a1, HbA.sub.1a2 and HbA.sub.1c are fructose diphosphate, glucose-6-phosphate and 1-deoxyfructose, respectively. The sugar attached to valine in HbA.sub.1b is not known.
About 4 to 5% of the total hemoglobin is HbA.sub.1c in normal persons, but the amount is substantially higher in diabetes, generally being about 8 to 10%, but sometimes as high as 20%. Also, its concentration varies widely in relation to diabetic control.
HbA.sub.1c is the most frequently measured glycosylated protein for clinical purposes, and a number of tests have been developed to measure it. See, e.g., Furth, Analytical Biochemistry, 175, 347-60 (1988); Peacock, J. Clin. Pathol., 37, 841-51 (1984); Miedema and Casparie, Ann. Clin. Biochem., 21, 2-15 (1984); and U.S. Pat. No. 4,629,692. Glycohemoglobin assays used routinely in clinical laboratories include: (1) affinity chromatography using m-aminophenylboronate columns; (2) cation exchange chromatography; (3) electrophoresis; and (4) isoelectric focusing. The most commonly used clinical methods are cation exchange chromatography for HbA.sub.1c and affinity chromatography for glycosylated hemoglobin. See Bodor et al., Clin. Chem., 38, 2414-2418 (1992).
Each of the four measurement techniques that are currently in clinical use suffers from one or more of the following disadvantages: relatively high cost per sample measured; lack of specificity in the analyte measured; sensitivity to slight variations in conditions such as ionic strength, pH and temperature; difficulty in standardization; lack of reproducibility; being time consuming and labor intensive; inability to automate; and difficulty in assaying many samples simultaneously. See U.S. Pat. No. 4,629,692; Furth, Analytical Biochemistry, 175, 347-60 (1988); Peacock, J. Clin. Pathol., 37, 841-51 (1984).
Immunoassays for measuring glycosylated proteins, particularly HbA.sub.1c, are known. These various immunoassay techniques, and the antigens and antibodies used in them, will now be discussed.
First, European Patent Application No. 201,187 describes the preparation of monoclonal antibodies using purified HbA.sub.1c. In particular, the application reports the preparation of monoclonal antibodies that preferentially bind to the glycated amino groups of hemoglobin, such as that of the N-terminal valine residues of the HbA.sub.1c beta chains. In particular, a glycated heptapeptide whose sequence corresponds to the sequence of the N-terminus of the beta chain of HbA.sub.1c inhibited the binding of one of these monoclonal antibodies to HbA.sub.1c, whereas the non-glycated heptapeptide and the reduced glycated heptapeptide did not inhibit this binding. The patent application teaches that these monoclonal antibodies can be used in known immunoassay techniques to quantitate HbA.sub.1c. It should be noted that only 2 out of 320 hybridomas produced monoclonal antibody preferentially reactive with HbA.sub.1c, suggesting that little antibody specific for HbA.sub.1c is normally produced by immunized animals.
U.S. Pat. No. 4,629,692 (Dean) teaches an immunoassay method for determining total non-enzymatically glycosylated proteins and protein fragments in a biological fluid. The immunoassay method employs an antibody which selectively recognizes and binds Amadori-rearranged glucose residues (i.e., 1-deoxy-D-fructosyl residues). The immunogen used to prepare the antibody is Amadori-rearranged glucose covalently bound to a carrier molecule. Polylysine is the preferred carrier, and the Amadori-rearranged glucose is preferably attached to the epsilon amino groups of the lysine residues. The immunogen is prepared by glycosylating the carrier non-enzymatically in vitro. A linker may be used between the Amadori-rearranged glucose and the carrier. The preferred linker is lysine. Other linkers including lysine analogs and amino-functionalized amino acids such as ornithine and hydroxylysine may be used.
The antibody prepared as described above can be used in a conventional immunoassay to quantitate total glycosylated proteins in a biological fluid such as serum. To determine the level of a specific glycosylated protein, such as HbA.sub.1c, it must first be separated from the other non-enzymatically glycosylated proteins. For instance, the Dean patent teaches that HbA.sub.1c can be separated by phenylboronate affinity chromatography and then quantitated using the disclosed antibody.
U.S. Pat. No. 4,658,022 (the '022 patent) teaches the preparation of antibodies to a linear peptide epitope of a protein. The linear peptide epitope comprises from 2 to 15 amino acids of any portion (N-terminal, C-terminal, or other portion) of the protein's sequence and may be modified with non-peptide groups such as carbohydrates. The linear peptide epitope is coupled to an immunogenic carrier for purposes of immunizing an animal. To perform the immunoassay, the antibodies are contacted with the protein which has been denatured sufficiently to expose, or increase the exposure of, the linear peptide epitope used for the preparation of the antibody. The use of monoclonal antibodies is preferred.
The '022 patent and U.S. Pat. No. 4,647,654 (the '654 patent) teach the use, in the system described above, of a glycosylated peptide containing at least 2 amino acids, preferably 5 to 15 amino acids, of the N-terminal sequence of hemoglobin coupled to an immunogenic carrier to prepare an antibody. The '654 patent teaches that a linker which optimizes antigenicity and coupling properties may be used between the peptide epitope and the carrier. The linker may comprise one or more amino acids not found in the normal sequence of hemoglobin. Both patents teach that monoclonal antibodies can be produced in this manner which are specific for the glycosylated synthetic peptide and the corresponding epitope on the HbA.sub.1c molecule when the HbA.sub.1c molecule is denatured to expose the epitope0 These antibodies do not cross-react with HbA.sub.o or with non-glycosylated peptides. The immunoassays used are conventional, except for the denaturation of hemoglobin. It should be noted that only 9 out of 200 hybridomas produced monoclonal antibody preferentially reactive with HbA.sub.1c, suggesting that little antibody specific for HbA.sub.1c is normally produced by immunized animals.
U.S. Patent No. 4,478,744 (Mezei et al.) teaches the preparation of antibodies to a protein using a peptide antigen, the amino acid sequence of which corresponds to a portion of the amino acid sequence of the protein. With respect to hemoglobin, the patent teaches the preparation of antibodies to glycosylated hemoglobin, specifically HbA.sub.1c, using a peptide consisting of 4 to 10, preferably 7, amino acids, the sequence of which corresponds to the N-terminal sequence of the beta chain of hemoglobin. The peptide is glycosylated before or after being coupled to an immunogenic carrier protein or polypeptide. The peptide-carrier combination is used to immunize an animal, preferably one that does not normally produce HbA.sub.1c. Mezei et al. teaches that the resultant antibodies are specific for HbA.sub.1c and may be used in conventional immunoassays to quantitate HbA.sub.1c. However, the '654 patent discussed above describes experiments showing that a polyclonal sheep antiserum produced according to the Mezei et al. method has no detectable specificity for HbA.sub.1c in an ELISA assay even when affinity purified (see Example 8 of the '654 patent). Also see, European Patent Application 201,187 discussed above.
U.S. Pat. No. 4,247,533 (Cerami et al.) and Javid et al., British Journal of Haematology, 38, 329 (1978) teach the preparation of antibodies to HbA.sub.1c. The antibodies are produced by immunizing an animal, preferably one that does not normally form HbA.sub.1c, with column purified human HbA.sub.1c. The resulting antibody reacted equally well with HbA.sub.o and HbA.sub.1c and was, therefore, absorbed repeatedly with HbA.sub.o. The absorbed antibody clearly distinguished HbA.sub.o from its glycosylated derivatives, but still cross-reacted slightly with human HbA.sub.1a and HbA.sub.1b and with dog and mouse HbA.sub.1c. It also showed strikingly less reactivity with NaBH.sub.4 reduced HbA.sub.1c than with unreduced HbA.sub.1c. Also, certain reduced glycodipeptides, including reduced glycosylated valyl-histidine, failed to inhibit the reaction of the absorbed antibody with HbA.sub.1c. The absorbed antibody was of low affinity and low titer. To overcome this problem, a specially modified radioimmunoassay (RIA) was employed. The '654 patent discussed above teaches that the reproducibility of this method is open to question (see column 2 , lines 38-41, of the '654 patent).
Curriss and Witztum, J. Clin. Invest., 72, 1427 (1983) describes the generation and characterization of six murine monoclonal antibodies that bind reduced glycosylated human plasma lipoproteins, but do not react with nonglycosylated or unreduced glycosylated plasma lipoproteins. The antibodies were prepared by immunizing mice with three injections of homologous low density lipoprotein (LDL) reductively glycosylated in the presence of glucose and sodium cyanoborohydride, followed by one injection of reductively glycosylated human LDL just before harvesting spleen cells to prepare the hybridomas. In competitive inhibition RIA, the dominant epitope recognized by these antibodies on reduced glycosylated LDL was identified as glucitol-lysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine (glucitol-lysine completely inhibited the binding of each of the antibodies to reduced glysosylated LDL). Each of the six antibodies reacted with all reduced glycosylated proteins studied, including high density lipoprotein, albumin, hemoglobin and transferrin. The antibodies were also capable of identifying and quantitating glucitol-lysine residues on total plasma proteins and isolated lipoproteins of normal and diabetic individuals after reduction of the proteins with NaBH.sub.4.
Witztum et al., Proc. Natl. Acad. Sci. USA, 80, 2757 (1983) describes the preparation of polyclonal antibodies by immunizing guinea pigs with homologous glycosylated or reductively glycosylated LDL. Immunization with reductively glycosylated LDL produced a high-titered antiserum that reacted with reductively glycosylated guinea pig LDL, but not with unreduced glycosylated LDL. Glucitol-lysine was a highly effective inhibitor of the binding of this antibody to reductively glycosylated LDL, as were other reductively glycosylated human proteins, including hemoglobin, albumin and transferrin. LDL glycosylated in the absence of a reducing agent was also immunogenic, although the antiserum was of lower titer and affinity. Homologous reductively glycosylated albumin was also immunogenic, and immunization with this compound produced an antiserum that reacted with reductively glycosylated albumin, but not with unreduced glycosylated albumin or reductively glycosylated LDL. All antibody activities were measured in a solid-phase RIA.
Nakayama et al., Clinica Chimica Acta, 158, 293-99 (1986) describes an RIA for glycated human serum protein using antiserum obtained by immunizing guinea pigs with reductively glycated human albumin. The antiserum was affinity absorbed on columns of native human serum albumin, and the absorbed antiserum recognized reductively glycated albumin and glucitollysine, but not non-reductively glycated albumin, native human albumin, lysine, sorbitol or mannitol. The antiserum was also capable of identifying and quantitating native human serum albumin and nonreductively glycated albumin after reduction of the protein with NaBH.sub.4.
Go et al., Clinical Chimica Acta, 163, 63-73 (1987) reports the development of an enzyme linked immunosorbent assay (ELISA) for glycosylated proteins using a polyclonal antiserum which was prepared using reductively glycosylated homologous high and low density lipoproteins (HDL and LDL) as the immunogen. The article teaches that the antiserum is specific for the glucose-lysine bond, and that it recognizes, in a dose-dependent manner, all reduced glycosylated proteins tested, including albumin, fibrinogen, LDL, HDL, polylysine and hemoglobin. The antiserum had no affinity for native proteins or for unreduced glycosylated proteins. The ELISA assay is reported to be sensitive and capable of measuring a large number of samples in a relatively short period of time, but the article teaches that the conditions of the assay are critical and that major deviations from the described protocol lead to loss in sensitivity and reproducibility. See page 66 of Go et al.